Accurate polynucleotide sequence determination is critically important in many applications. For example, comprehensive definition of an individual's genetic profile requires that long stretches of chromosomal DNA are determined and compared against databases of known sequences. The results can establish profiles of predispositions or susceptibilities to provide medical insights. Likewise, tumor profiling also can require accurate sequence determination to establish the efficacy of a drug treatment regimen before treatment has begun. As well, identifying bacterial, viral, or other pathogens from nucleic acid databases also can depend on accurate sequencing results.
Stretches of more than one of the same base along a strand of nucleic acid are among the factors confounding accurate sequence determination. These “homopolymer” stretches can be overlooked by some sequencing approaches, such that a single base will be detected when multiples actually are present. Some sequencing methods further can experience “phasing” issues that can be exacerbated by the homopolymer stretches. As a consequence of phasing, sequence determination downstream of a homopolymer stretch can be rendered ambiguous.
Different approaches have been developed to address and resolve homopolymer sequencing issues. Reversible terminator nucleotides have been employed to ensure that only a single nucleotide is enzymatically incorporated into a growing primer. While effective, follow-on steps can be required to remove the chemical terminator moiety from the primer before the next round of template-dependent incorporation can occur. Other approaches have involved measuring the amplitude of incorporation signals using only one species of nucleotide at a time. Unfortunately, this approach imposes limits on the length of sequence that can be determined. Thus, there remains a need for new approaches that can be used for sequencing nucleic acids, including accurate sequencing through homopolymer stretches within a nucleic acid strand.